Pulmonary microvascular endothelium forms a restrictive barrier to allow proper gas exchange. Inflammatory mediators and vascular permeability-increasing compounds cause retraction of cell borders and inter-endothelial gaps by reorganizing the endothelial cytoskeleton, cell-cell, and cell matrix interactions. Inflammatory mediators induce two critical changes in the endothelial cell cytoskeleton, including reorganization of the cortical actin rim into stress fibers, and disassembly and reorganization of microtubules. Despite their importance in control of endothelial cell shape, relatively little is known about intracellular signals that control microtubule assembly and disassembly. Microtubule-associated proteins regulate microtubule dynamics. Tau is a major neuronal microtubule-associated protein that promotes assembly, stability, and bundling of axonal microtubules. Tau hyper-phosphorylation reduces Tau microtubule binding-ability and causes neurofibrillary tangle formation, a pathologic severity marker of Alzheimer's disease. Protein Kinase A contributes to neurofibrillary tangle formation by phosphorylating Tau at serines 214, 262, and 356. Just recently, non-neuronal Tau has been found to regulate microtubule dynamics in endothelium. In endothelial cells, soluble adenylyl cyclase activity has been associated with Tau serine 214 phosphorylation and microtubule reorganization, and preliminary data suggest that Tau serine 214 phosphorylation releases Tau from endothelial microtubules. Interestingly, bacteria have evolved toxicity mechanisms that insert soluble adenylyl cyclases into endothelial cells and increase permeability. Since multiple different kinases can phosphorylate Tau, the mechanism by which adenylyl cyclase activity induces Tau hyperphosphorylation and endothelial barrier disruption is unknown. It is similarly unclear whether bacterial adenylyl cyclases induce endothelial hyperpermeability by a Tau-dependent mechanism. Therefore, the present application tests the overall hypothesis that bacterial adenylyl cyclase toxins induce Tau serine-214 phosphorylation that promotes microtubule disassembly and increases microvascular endothelial permeability. Specific aims will test the related hypotheses that: [1] Bacterial soluble adenylyl cyclase toxins activate PKA that phosphorylates Tau serine 214; and [2] Phosphorylation of Tau ser-214 is sufficient to disassemble microtubules and increase permeability. Completion of these studies will not only impact the understanding of endothelial permeability and bacterial-induced acute lung injury, but will also be significant in the field of neurodegenerative diseases. In the present proposal, we will rigorously test whether bacterial adenylyl cyclases generate a cAMP signal that results in phosphorylation of Tau ser-214 sufficient to depolymerize microtubules and increase endothelial permeability, and as PKA phosphorylation of Tau is a known pathophysiological event in Alzheimer's disease, our studies will resolve a putative mechanism responsible for PKA activation that results in Tau hyperphosphorylation.